Fig. 1: ZAK induces EMT and stem cell-like properties in epithelial cell lines

a Changes in the expression of EMT markers upon ZAK overexpression. Top, western blot analysis of mesenchymal markers (vimentin, fibronectin, and N-cadherin) and epithelial markers (E-cadherin and occludin) in stable cell lines expressing ZAK, other candidate EMT kinases, positive kinase controls (MET and FYN), and empty vector control (EV). Bottom, RT-PCR analysis of ZAK, vimentin, and E-caderin (CDH1) expression. Shown on the Y-axis is log10-fold change of mRNA level induced by ZAK vs. vector control. Error bars denote SD from triplicate. b Immunofluorescence staining patterns of vimentin and E-cadherin in HMLE and PC3 cells expressing EV or ZAK. Cell nuclei were stained with DRAQ5. Scale bars, 50 μm. c ZAK promoted migration as determined by Boyden chamber assay, showing representative photos (top) and quantification (bottom) of migration. Data are represented as mean ± SD of triplicate experiments. ***P < 0.001 (Student’s t-test) d Proliferation curves of epithelial cell lines expressing ZAK or EV grown in regular media or growth factor reduced medium. GF growth factors. The number of viable cells was measured by AlamarBlue assay at different time points and data are expressed as viable cell number and fold changes from that at Day 1. Mean values ± SD from three independent experiments are shown. *P < 0.05, **P < 0.01 and ***P < 0.001 (Two-way ANOVA followed by Bonferroni post-tests). e Dose-response curves of indicated cell lines treated with chemotherapeutic drugs. IC50 values were obtained using logistic nonlinear regression analyzing model of MicroCal Origin 8.5 software. Error bars denote SD from two independent experiments, each done in quadruplicate. *P < 0.05 and ***P < 0.001 (Student’s t-test). f FACS analysis of CD44 and CD24 in HMLE-EV and HMLE-ZAK cells. The percentage of CD44^high/CD24^low stem-like subpopulation is indicated. g HMLE-ZAK cells generated more and bigger mammospheres than HMLE-EV control cells, showing representative photos (top) and quantification of numbers and diameters of mammospheres from three independent experiments (bottom). Data are represented as mean ± SD of triplicate experiments. Statistical analysis was done using Student’s t-test. h HMLE cells expressing ZAK gained mesenchymal stem cell-like capabilities for multilineage differentiation. Following culture in osteoblastic differentiation media, cells were tested for alkaline phosphatase (AP) activity, or analyzed by silver nitrate (Von-Kossa) staining and alizarin red S staining to determine mineral deposition and calcium deposition. Following culture in adipogenic differentiation media, cells were stained with oil red dye to detect oil droplets formation. Additional data related to Fig. 1 are in Supplementary Figure S1